Stem cells (both normal and cancerous) are defined by their ability to self-renew, in order to maintain their numbers, and their ability to differentiate into distinct cell types. Because of these competing functions, the genome of stem cells must be uniquely regulated ~ stem cells must stably maintain their gene expression pattem during self-renewal, but must be flexible enough to drastically alter their gene expression pattern during differentiation. The gene expression patterns of stem cells are regulated by transcription factors and Chromatin regulators expressed in the cell. While transcription factors that control stem cell self-renewal vary considerably between different types of stem cells, there is increasing evidence that chromatin regulators of self-renewal function more broadly. Recently we performed an RNAi screen for chromatin regulators with mportant functions in embryonic stem cells and found 68 genes with an array of knockdown phenotypes. We will now screen these factors for function in a different stem cell type, cancer stem cells. Using both mouse models and human cancer cell lines, we will identify chromatin regulators of cancer stem cells, with the goal of identifying novel targets for more effective therapies that target the cancer stem cells. We will characterize the molecular functions of chromatin regulators identified in the screen to gain a greater understanding of the roles of chromatin regulation in cancer stem cell self-renewal. Next, we will identify functional interactions among transcription factors and chromatin regulators in embryonic stem cells. Although much is known about the network of transcription factors that function in embryonic stem cells, the mechanisms by which they interact with chromatin regulators to control gene expression are largely unknown. By examination of the global effects of knocking down chromatin regulators and pluripotency transcription factors (alone or in combination) on gene expression, we hope to identify novel functional interactions important for stem cell self-renewal. Subsequently, the mechanisms by which groups of transcription or chromatin regulators function together will be examined. These data should lead to a greater understanding of stem cell gene regulation, which will ultimately be useful for future stem cell-derived therapies.